Interleukin (IL)-13 promoter polymorphisms (-7402 T/G and -4729G/A) condition susceptibility to pediatric severe malarial anemia but not circulating IL-13 levels

Plasmodium malaria is one of the leading causes of morbidity and mortality in the world [1]. World Health Organization (WHO) estimated that 216 million malaria episodes occurred in the year 2010, resulting in approximately 655,000 deaths, of which 91% occurred in the sub-Saharan Africa [2]. Children under five years of age bear the greatest disease burden, with a global malaria related mortality of 86% documented in 2010 [2]. In malaria endemic regions of sub-Saharan Africa, greater than 90% of malaria-related deaths are attributable to Plasmodium falciparum, the most virulent of the five malaria parasite species known to cause human malaria [3]. The clinical spectrum of severe falciparum malaria often ranges from mild symptoms to life threatening complications which include, but are not limited to, severe anemia, respiratory distress, cerebral malaria (CM), hyperparasitemia, metabolic acidosis, kidney failure, and hypoglycemia [4]. In western Kenya, a falciparum malaria holoendemic region, the most common clinical manifestation of severe malaria is severe malarial anemia [SMA (Hb < 6.0 g/dL)], with any density parasitemia [5, 6], with respiratory distress, hypoglycemia and CM reported only in rare cases [7‐9]. In addition, the etiology of SMA is complicated by the presence of co-infections and by host genetic factors [9‐14]. Furthermore, additional studies have demonstrated that immunity against SMA is characterized by dysregulation of innate types 1 and 2 inflammatory mediators, mainly cytokines, chemokines and effector molecules [15‐17].

Among the inflammatory mediators implicated in malaria pathogenesis is interleukin (IL)-13, a type 2 cytokine that shares structural and functional characteristics with IL-4 [18, 19]. IL-13 is a pleiotropic cytokine predominantly produced by activated T lymphocytes, mast cells, basophils, natural killer (NK) cells and dendritic cells [20]. It functions as an immune modulator on a wide range of cell types, including macrophages, NK cells, fibroblasts, eosinophils, airway smooth muscle cells, and endothelial cells [20]. Elevated levels of IL-13 have been implicated in allergic responses and in parasitic infections [21‐23]. Recently, IL-13 has emerged as a negative predictor of hemoglobin (Hb) levels for pediatric malarial anemia [17].

The human IL-13 gene has been mapped to the 5q31-q33 region, located on chromosome 5, which contains additional genes encoding cytokines, including IL-3, IL-4, IL-5, IL-9, and IL-12B [24‐28]. Polymorphisms in the promoters of genes coding for these cytokines have been shown to alter susceptibility to infectious diseases [11, 25, 29, 30]. Several polymorphic variants identified in the IL-13 gene region [25, 30, 31] have been associated with various diseases including allergic rhinitis [32], asthma [33], atopic dermatitis [22], and schistosomiasis [25]. In addition, studies in Thai adults demonstrated that a single nucleotide polymorphism (SNP) in the IL-13 promoter (IL-13 -1055C > T) is associated with severe malaria [31]. However, to date, no studies have reported the associations between IL-13 promoter variants -7402G/T (rs7719175) and -4729G/A- (rs3091307), their haplotype constructs and SMA in pediatric populations resident in P. falcipuram holoendemic transmission region such as in western Kenya. The IL-13 promoter variants -7402G/T (rs7719175) and -4729G/A- (rs3091307) were selected based on the mutant allele frequency ~10% in reference African Yoruba population (http://​hapmap.​ncbi.​nlm.​nih.​gov/​downloads/​frequencies/​). Consequently, the current study investigated the associations between two IL-13 promoter variants, haplotypes and susceptibility to SMA in a phenotypically well-defined cohort of children aged 3–36 months presenting with clinical symptoms of malaria in a rural setting of western Kenya.

To the best of our knowledge, this is the first study to report on the association between the IL-13 promoter polymorphisms (-7402 T/G and -4729G/A) and susceptibility to SMA in well-defined phenotypic pediatric populations resident in P. falciparum holoendemic transmission region of western Kenya. Our results, obtained from this cross-sectional study of a cohort of parasitemic children (n = 387) aged 3–36 months in this P. falciparum holoendemic region, revealed that none of the individual IL-13 genotypes was associated with susceptibility to SMA. Further analyses, however, revealed that the individuals with the -7402 T/-4729A (TA) haplotype were significantly over-represented in the SMA group relative to non-SMA. Carriage of the TA haplotype was associated with susceptibility to SMA, confirming our findings that this haplotype could confer genetic predisposition to SMA in children naturally exposed to P. falciparum infection. Frequencies of the TT genotype at the -7402 T/G genotypes was higher in our population relative to that of the reference Yoruba population [39], showing that this locus may be undergoing some selective pressure in our population either as a result of disease or any other unidentified selection. In addition, there is likely to be additional SNPs in linkage disequilibrium with the IL-13 variants that may in turn lead to the observed association between the studied SNPs and SMA. It would be important to map additional SNPs in the IL-13 loci and adjacent genes in order to further identify variants driven by disease-related selections.

Haplotypes are important markers for identifying associations with disease outcomes that are not identifiable with SNPs [11]. Previous studies, using different disease (other than SMA) models showed associations between individual (-7402 T/G and -4729G/A) and parasitic infections and allergic responses [25, 30]. In the current study, carriage of the -7402 T/-4729A (TA) haplotype was associated with increased susceptibility to SMA. Consistent with the finding that TA haplotype is associated with susceptibility to SMA, the presence of the haplotype was over-represented in SMA relative to non-SMA group. Even though the mechanism through which carriage of the TA haplotype increases susceptibility to SMA remain to be determined in our laboratory, we hypothesize that the presence of the TA haplotype may be promoting non-erythropoietic responses in children with falciparum malaria through alteration of IL-13 promoter binding of transcription factors to the IL-13 promoter region. Previous studies have demonstrated that changes that affect IL-13 and IL-13Rα1 at the D1 domain of the type II IL-4 receptor [18], leading to alterations in signaling for production of IL-13 via activation of the transcription factor signal transducer and activator of transcription 6 (STAT6) may affect erythropoiesis [40]. Other studies have also shown that the IL-13Rα1 receptor forms a heterodimer with IL-4Rα which provide signaling pathways for both IL-13 and IL-4 [41‐43]. IL-4 has further been shown to be an erythropoietic inhibitor whose elevated production can aggravate severe malaria [44]. As such, the presence of the TA haplotype could be altering the gene-gene interactions of IL-13 and IL-13Rα1, thereby promoting enhanced IL-4 production leading to inhibition of erythropoiesis in children with malaria. Since there were no relationships between individual IL-13 genotypes and haplotypes and circulating IL-13 levels in the current study, there is likely to be additional regulators either upstream or downstream of the IL-13 promoter that may alter the IL-13 signaling pathway during disease. Our laboratory is currently investigating the possibility of these additional regulators in functionally altering a milieu of immune mediators during malaria disease pathogenesis in children.

Previous studies have demonstrated that increased IL-13 production exacerbate infectious diseases and allergic disorders [25, 30, 45, 46], alter immune responses in malaria [47‐49], and is potentially a negative predictor of Hb levels in children with malarial disease [17]. Even though a previous study demonstrated that variants within the IL-13 (-1055C/T) promoter was associated with severe malaria in an adult Thai population [31], likely through alterations in the binding of transcription factor (NF-AT) on the IL-13 promoter region thereby leading to differential production of IL-13 levels, no study to date had determined the role of -7402 T/G and -4729G/A in altering IL-13 production in children with malaria. We demonstrate that these variants (genotypes and haplotypes) do not independently alter IL-13 circulating levels in our pediatric population. In addition, contrary to previous findings [25, 47‐49], circulating IL-13 levels did not significantly differ between the SMA and non-SMA in our cohort. Even though previous studies have additionally demonstrated that IL-13 is involved in alternative activation of macrophages, a mechanism specialized for defense against extracellular pathogens [50], it is important to note that P. falciparum is an obligatory intracellular pathogen [51], and thus, the defense mechanism provided by alternative activation of macrophages may not absolutely aid in defense against it.

Furthermore, differences in the current results versus previous findings [25, 47‐49] could be due to geographical and demographic differences which may concomitantly be accompanied by various selective pressure. For example, the current pediatric population is naturally exposed to additional conditions such as other parasitic infections, which may independently alter IL-13 production [52, 53]. These other parasitic infections may (in the presence or absence of P. falciparum malaria) complicate IL-13 production in relation to other immune mediators [52, 53]. It is also important to note that infectious diseases occur through complex and multifactorial mechanisms [10, 22, 36, 47, 53, 54], and as such, in populations in which there is likely to be multiple co-infections, it would be critical to delineate and stratify them into those with clearly defined disease status and carry out a comprehensive analyses of an all inclusive immune mediator analyses to shed more light into those immune mediators that significantly alter malaria disease outcomes.

The current study reports for the first time, the association between the IL-13 promoter polymorphisms and haplotypes (IL-13 -7402 T < G/ and -4729G/A) and SMA in parasitemic children below 3 years of age in a P. falciparum malaria holoendemic transmission area of western Kenya. Even though the IL-13 promoter genotypes and haplotypes did not independently alter circulating IL-13 levels, carriage of the -7402 T/-4729A (TA) haplotype appeared to be genetically predisposed to development of SMA once the children acquired P. falciparum malaria. It would be important to carry out additional studies in independent populations experiencing different malaria disease outcome(s) to validate these findings.

The study was approved by the Ethics Review Committee of the Kenya Medical Research Institute. Informed written consent was obtained from the parent or legal guardian of all children participating in the study.

Department of Biomedical Sciences and Technology, Maseno University, Maseno, Kenya (WAO and EOM); Lecturer, Department of Medical Biochemistry, Maseno University, Maseno, Kenya (WO and SBO); Lecturer, Department of Medical Laboratory Sciences, School of Health Sciences, Masinde Muliro University of Science and Technology, Kakamega, Kenya (ER); Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya (JMV); Research Officer, University of New Mexico/KEMRI Laboratories of Parasitic and Viral Diseases, Centre for Global Health Research, Kenya Medical Research Institute, Kisumu, Kenya (JMO); Professor and Director, Center for Global Health, Department of Internal Medicine, University of New Mexico, Health Sciences Centre, Albuquerque, NM, USA (DJP); Associate Professor, Department of Biomedical Sciences and Technology, and Director, Research, Publications and Consultancies, Maseno University, Maseno, Kenya (CO).